Nerve growth factor (NGF) is the first neurotrophin identified in 1951, and is an important secretory protein involved in the development and survival of peripheral and central neurons. It consists of 118 amino acids, has a molecular weight of 13 kDa, and has S—S bonds at 3 positions in a molecule. BDNF, NT-3 and NT-4/5 are present in the family protein, which are structurally well conserved and form a homodimer by a noncovalent bond. It has a β sheet structure facing 3 different directions, and is considered to be dimerized in this part. It also has four loop structures with low homology among families, and these parts are considered to define specificity to receptors.
As NGF receptors, tyrosine kinase-type receptor TrkA with high affinity and p75 with low affinity which belongs to a tumor necrosis factor receptor superfamily are known. These receptors act as a homodimer or heterodimer and are deeply involved in the development and maintenance of the nervous system. TrkA is a single-pass transmembrane receptor and has a tyrosine kinase structure in the intracellular domain. When NGF is bound, tyrosine phosphorylation occurs, the signal is transmitted to the downstream, and promotion of differentiation and survival maintenance of the cell occur.
As family receptors of TrkA, TrkB and TrkC are known. TrkB is bound to BDNF and NT-4/5, and TrkC is bound to NT-3. p75 shows lower ligand specificity as compared to TrkA and is also bound to BDNF, NT-3 and NT-4/5 besides NGF. While p75 is a single-pass transmembrane receptor, it does not have a tyrosine kinase domain on the cytoplasmic side. Like TrkA, it is expressed not only in nerve cells but also in non-nerve cells. This receptor is known to be involved in the promotion of differentiation and survival maintenance of the cell, as well as related to the induction of apoptosis and cell migration. The results of crystal structure analysis have suggested that an NGF homodimer binds to TrkA at 2:2 and to p75 at 2:1. An NGF homodimer sometimes binds to a heterodimer of TrkA and p75.
NGF is produced by Schwann cell, keratinized cell, bronchial epithelial cell, fibroblast, T lymphocyte, macrophage, mast cell, B lymphocyte, keratinocyte, smooth muscle cell, renal glomerular cell, skeletal muscle cell and the like. On the other hand, TrkA is known to be expressed in nerve cell, as well as monocyte, T lymphocyte, B lymphocyte and mast cell other than nerve cell. Similarly, p75 is expressed in nerve cell as well as non-nerve cells.
It is well known that NGF plays a key role in the nervous system. It has been clarified that NGF has an action to maintain survival of cholinergic neuron and is considered to be related in some way to Alzheimer's disease. In addition, since intracerebral administration of NGF improves memory disorders of old rats, it is also expected as a therapeutic drug for senile dementia.
It has been found that NGF also acts on the tissues and cells other than the nervous system, and involved in the body's defense and tissue repair process. For example, it is known that administration of NGF to an animal increases blood vessel permeability, enhances immune responses of T cell and B cell, induces differentiation of lymphocytes, induces growth of mast cells, induces release of various cytokines from mast cells and the like.
NGF is related to inflammation, and increased expression of NGF has been observed in patients with inflammatory diseases and inflammatory animal models. Systemic lupus erythematosus, multiple sclerosis, psoriasis, arthritis, interstitial cystitis, asthma and the like are the examples thereof. It has been reported that the synovial fluid of patients with rheumatoid arthritis shows higher NGF concentration. In addition, increased NGF expression in rheumatoid arthritis model rats, and increase in mast cells and increased NGF expression in arthritis model mouse have been reported.
NGF is deeply involved in pain. When NGF is subcutaneously administered to human, a deep pain such as muscular pain continues for several days, and hyperalgesia of the injection site occurs. NGF knockout mouse and TrkA knockout mouse lacks unmyelinated nerve and do not feel pain. When NGF is intraperitoneally administered at 1 mg/kg to a mature rat, hyperalgesia against noxious heat and mechanical stimuli occurs. NGF transgenic mouse shows hyperalgesia unaccompanied by inflammatory conditions. In addition, it is known that the TrkA gene of patients with congenital insensitivity to pain with anhidrosis (CIPA) has abnormality, and pain sensation decreases when NGF gene has abnormality.
From the above, an NGF inhibitor can be used as a therapeutic drug for pain such as nociceptive pain, inflammatory pain, neuropathic pain, carcinomatous pain, fibromyalgia pain and the like. A combination therapy of NGF antibody and NSAID (Patent reference 1), a combination therapy of NGF antibody and opioid analgesic (Patent reference 2), a treatment method of postsurgical pain using an NGF antibody (Patent reference 3, Patent reference 4), a treatment method of pain of bone cancer using an NGF antibody (Patent reference 5), and a treatment method of pain of osteoarthritis using an NGF antibody (Patent reference 6) have been reported.
Tanezumab (PF-4383119 or RN624) is an antibody against NGF, shows effect in pain model experiment using an osteoarthritis animal model, and is currently under clinical trial. While the presence or absence of inhibitory activity of NGF and NGF receptor is unknown, there is a report relating to natural RNA that binds to NGF (non-patent document 1).
In recent years, applications of RNA aptamers to medicaments, diagnostic agents, and test drugs have been drawing attention; some RNA aptamers have already been in clinical study stage or in practical use. In December 2004, the world's first RNA aptamer drug, Macugen, was approved as a therapeutic drug for age-related macular degeneration in the US. An RNA aptamer refers to an RNA that binds specifically to a target molecule such as a protein, and can be prepared using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Patent references 7-9). In the SELEX, an RNA that binds specifically to a target molecule is selected from an RNA pool with about 1014 different nucleotide sequences. The RNA structure used has a random sequence of about 40 residues, which is flanked by primer sequences. This RNA pool is allowed to be assembled with a target substance, and only the RNA that has bound to the target substance is collected using a filter and the like. The RNA collected is amplified by RT-PCR, and this is used as a template for the next round. By repeating this operation about 10 times, an RNA aptamer that binds specifically to the target substance can be acquired.
Aptamer drugs, like antibody drugs, can target extracellular factors. With reference to many scientific papers and other reference materials in the public domain, aptamer drugs are judged to potentially surpass antibody drugs in some aspects. For example, aptamers often show higher binding force and higher specificity than do antibodies. Aptamers are unlikely to undergo immune elimination, and adverse reactions characteristic of antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), are unlikely to occur with the use of aptamers. From the aspect of delivery, since aptamers are about 1/10 of antibody in size, delivery of a drug to the object site is easier. Since aptamers are produced by chemical synthesis, various modifications can be made easily, reduction of cost by large-scale production is possible. Meanwhile, the blood half-lives of aptamers are generally shorter than those of antibodies; however, this property is sometimes advantageous in view of toxicity. These facts lead to the conclusion that even when the same molecule is targeted, aptamer drugs potentially surpass antibody drugs.
The present inventors have produced, in PCT/JP09/066,457, an aptamer which binds to NGF and inhibits binding of NGF and an NGF receptor, and found that the aptamer inhibits a neurite outgrowth activity of NGF. Patent document 10 describes an aptamer to NGF, which is obtained by automated SELEX, and patent document 11 describes an altered product and a modified product of the aptamer obtained in patent document 10.